1) Our objective was to determine how Wnt signaling, which antagonizes neural development, is prevented in the anterior neurogenic ectoderm, termed the animal pole domain or APD. At least two genes encoding proteins that can antagonize Wnt (sFRP1/5 and Dkk1) are expressed in this region. Mis-expression of each can prevent Wnt-dependent endomesoderm development and prevents clearance of the animal pole domain factors from the lateral ectoderm. Loss of Dkk1 allows Wnt signaling to eliminate the entire APD. Unexpectedly, loss of sFRP1/5 leads to a larger APD. Thus, sFRPs role is not yet clear, but it may indirectly increase the domain of Dkk1 activity. Several Wnt receptors are expressed in the animal hemisphere when the APD is differentiating at the animal pole. One of these, Fzl1/2/7, is required for APD development while the second, Fzl5/8, antagonizes it. Our results support a model in which Dkk1s antagonism of Fz5/8 protects the APD. Manuscript in preparation. 2) De novo neurogenesis in the foregut. (25%) (Zheng Wei, Lynne Angerer) We made the surprising discovery that pharyngeal neurons of sea urchin embryos develop de novo from the endoderm in the foregut through the activity of the transcription factors, Six3, Nkx3-2 and Brn1/2/4. This result is entirely unexpected because a fundamental concept in developmental biology is that nerves form from ectoderm. We ruled out migration of ectodermal cells to the pharynx by tracking all presumptive ectoderm cells with the photo-activatable protein, KikGR. We observed that neurons appear in the foregut before the stomodeal ectoderm joins the foregut and in exogastrulae. We found that Nkx3-2 is co-expressed with synaptotagmin B, which marks differentiating neurons, in several cells in the foregut of gastrulae, consistent with the dependence of these neurons on Nkx3-2. These and other results (Peter et al., 2010, Dev. Biol. 340: 188-199) suggest both endodermal and neural gene regulatory networks operate in foregut cell lineages before gastrulation. Manuscript submitted. 3) Mechanisms underlying endomesoderm segregation. (25%) (Adi Sethi, Lynne Angerer) We have determined that a major distinction between endoderm and mesoderm at late mesenchyme blastula/early gastrula stages is the presence and absence of TCF in endoderm and mesoderm nuclei, respectively. Since TCF is the binding partner of beta-catenin, it is a critical component of canonical Wnt signaling, which is required for endoderm development. In the endoderm, transcription factors that are constituents of the early endoderm network are required to maintain TCF within nuclei, whereas in the mesoderm, Notch signals are required to promote its export from nuclei. While differential regulation of nuclear TCF levels in endoderm and mesoderm is likely to help establish stable regulatory states in these tissues, it is not among the first specification steps. Those include Notch-dependent activation of genes required for mesoderm-specific gene expression programs and down regulation of factors supporting the endoderm program during blastula/early mesenchyme stages. One of these endoderm factors, although known to depend on canonical Wnt signaling, also appears to be especially important in supporting it during mid-mesenchyme blastula stages. In addition, it supports expression of other endoderm regulatory proteins shortly after its appearance in presumptive endoderm, and these functions surprisingly depend on Notch signaling. Further studies are underway to determine how Notch signals in endoderm cells affect canonical Wnt signaling, the operation of the endoderm gene regulatory network and the stabilization of TCF in endodermal nuclei. Manuscript in preparation. 4) Dopaminergic neurons regulate the embryos response to food density (25%) (Diane Adams, Lynne Angerer) Previous work with pharmacological inhibitors of dopamine receptor function suggested that dopamine signaling was involved in the embryos response to food density. We have confirmed this hypothesis by perturbing this pathway at the level of dopamine production or by eliminating a dopamine D2 receptor. A major part of this response is to divert energy from maternal lipid stores to lengthen arms in order to increase feeding rate. We have initiated studies on dopaminergic neurogenesis and defined a new cell type with unique markers, including the dopamine biosynthesis enzyme dopa decarboxylase. Because some of these cells are positioned near the points of skeletal growth, they are excellent candidates for mediating the skeletal growth response. This mechanism is a novel developmental response of the embryo to its environment. Manuscript in preparation